Human cytomegalovirus (HCMV) is the largest and most complex human herpesvirus. The 250 kilobase linear dsDNA genome contains over 200 open reading frames and extracellular virus is composed of at least 35-40 structural proteins. Although significant effort has been directed towards understanding its complex transcriptional program, only limited information has been gathered on the intracellular assembly of enveloped virions. Envelopment is thought to occur in the cytoplasm by budding of tegument bound subviral particles into vesicles containing fully processed envelope glycoproteins. The envelope of HCMV consists of at least 6 unique glycoproteins, although the major envelope glycoprotein, glycoprotein B (gB), accounts for well over 50% of the envelope. Because gB appears to be the major structural element of the envelope, the assembly and envelopment of the viral particle is highly dependent on the structure and cellular localization of gB. The morphogenesis of the HCMV envelope will be analyzed by defining the intracellular folding, transport, and cytoplasmic localization of its major structural component, gB. Structures within gB which direct oligomerization and folding of the molecule and thus its transport from the ER will be determined. Mutations in various regions of the molecule including deletion of N-linked glycosylation sites and disulfide bonds will be made and the resulting phenotypic changes in its folding and transport characterized. The structural requirements for incorporation gB into cytoplasmic vesicles will be assayed by isolation and analysis of intracytoplasmic vesicles. The data, methodology, and reagents produced in these studies will be used to directly examine the effects of mutations in gB on the assembly of HCMV in infected cells. These later studies will be accomplished by the use of a gB deletion mutant and complementing cell lines constitutively expressing gB. The goal of these studies is to provide further understanding of the critical role of the major envelope protein of HCMV in envelope morphogenesis and virion assembly.